The ability to measure fluid velocity is important for realizing a full potential of micro- and nano-fluidics, especially for assays, where multiple screening events are required. Recent work has demonstrated that confinement of electrokinetic molecular transport in fluidic channels with transport limiting pore sizes of nanoscopic dimensions (approximately 100 nm) gives rise to unique molecular separation capabilities (references 1 and 2). Understanding fluid and molecular transport in nanoscopic channels is a tremendous challenge because of the lack of experimental methods that are available for interrogating the positions and trajectories of molecules at sub-wavelength dimensions. Silicon-based T-chips containing an array of parallel nanochannels can be used to study the electrokinetic transport of fluorescent dyes in nanochannels. Fluorescent imaging using confocal microscopy is an excellent method for the direct observation of molecules in chemical separation due to its high sensitivity (reference 3). While the details for the flow profiles in the individual nanochannels are below the resolution limit of optical microscopy, average velocities of dye fronts can be monitored and provide insight into the electrokinetic transport mechanisms in the nanochannels. Simultaneous transport of positive and negative dyes in the channels provides better understanding of the electrokinetic mechanism of separation.
Microscale flow visualization methods applied for the determination of fluid velocity in Microsystems is a well addressed. (references 4 through 9). The task of change detection is to determine how much and in what direction has a pixel changed. Velocity estimation methods can be roughly divided into two groups: particle and model-based spot tracking methods and image derivative based methods like optical flow. The types of movement generally studied by these methods usually involve discrete object or multiple objects that are changing their location within an image field of view. The particles are often added into the fluid, such that the transport is detected but not altered. Spot-tracking methods rely on accuracy of the segmentation of characteristics objects, such as particles or cells. No segmentation is needed for optical flow methods. The overall or “average” velocity describing all objects moving within a system of study is obtained by these methodologies. For movement of dye front within an array of nanochannels, the transport properties are quite different; namely, there are no discrete objects for which intensity profiles can be monitored as a function of time. The front moves as a plug profile in the array of nanochannel. With time, the nanochannels are getting filled up by the dye, which may or may not leave the entrance side of the nanochannels. Moreover, “average” velocity is not a useful parameter in studying separation of species moving with different velocities within micro- and nanochannels. For such type of movement, velocity calculation is done manually through plotting a large number of horizontal profiles within each confocal image from individual channels capturing only one type of fluorescing species (red or green) and determining the position of the dye front from those horizontal profiles. This is time-consuming and somewhat subjective procedure. The large number of horizontal profiles needs to be processed to obtain an accurate representation of average velocity of the dye front.
Nanofluidic arrays typically involve the use of very small sample volumes. This very small sample volume makes forming analyte “bands” and detection of individual molecular velocities by traditional means difficult. Furthermore, as with gels, electrophoretic (cross-reactive) analytes may require different analysis times and thus optimal placement relative to the array.